Vanilla aldehyde (4-hydroxy-3-methoxy-benzaldehyde), commonly referred to as vanillin, is the main fragrance ingredient of Vanilla planifolia Andreurs (Vanilla) which is prestigious as “the king of food-flavorings in the world”. Vanillin possesses a pure perfume of vanilla pods as well as an intense taste of sweet, creamy and cocoa characteristics and salubrious scent. Therefore, vanillin is an important broadspectrum top-grade flavoring in the world and widely used in foodstuff, beverage, tobacco, alcoholic, cosmetic, pharmaceutical and chemical industries.
At present, “artifical” or semi-synthetical vanillins are mostly derived from benzene-based petrochemical materials. For example, vanillin may be synthesized from guaiacol through Reimer-Tiemann reaction or chemically synthesized from lignin as starting material. Although these methods are simple and can be used for the large-scale production of cheap products, these methods have a disadvantage of causing environmental pollution. In addition, the starting materials employed in these methods such as guaiacol is toxic. Extraction from plant (vanilla pods) is another production method for vanillin. Such a method, however, is limited by climate and terrain conditions and the production yield is low (about 20 ton per year), resulting in a high price (1,200-4,000 $/kg). Along with an increased emphasis on the food safety by society, the consumers' demand for natural flavorings in foods has increased over the years. The legal definition of “natural flavor” in both the US and the major European countries includes products obtained by fermentation and enzymatic processes (EC (88/388) and FDA (CFR21)). This definition offers the flavor industry the chance of using biotechnology for the production of natural flavors. To meet the people's need for natural vanillin, methods for the production of vanillin using microbes or enzymes have been developed. In the May 2000 edition of “Chemistry in Britain”, pages 48-50, it is suggested that bio-conversion of ferulic acid to vanillin by liquid cultures of various fungi may be an economical route as well.
Ferulic acid, chemically similar to vanillin, is an important substrate for the whole transformation process from glucose to vanillin in plant, and therefore is considered as a promising precursor for vanillin. Gross et al. (U.S. Pat. No. 5,262,315) provides a process for producing vanillin by bioconversion of benzenoid precursors (the group consisting of vanillic acid and ferulic acid) by a basidiomycete fungus of the genus Pycnoporus, discloses that 0.3 g/L of ferulic acid is converted into 0.045 g/L of vanillin by P. cinnabarinus CNCMnDEG I-937 or CNCMnDEG I-938 with a molar conversion yield of 20.5%, wherein vanillic acid is transformed into vanillin with a conversion yield of 35.1%. Lesage-Meessen et al. (U.S. Pat. Nos. 5,866,380; 6,162,637) employs a two-step method using filamentous fungi to produce vanillin with an increased yield. The first step of the two-step method is to add ferulic acid to fermentation broth of microbial strain Asp. niger MIC 373, wherein ferulic acid is added in continuous fashion in the proportion of 430 mg/l per 24 hours to a final concentration of 5.05 g/L. After culturing for 15 days, the final concentration of vanillic acid in the culture is detected by HPLC to be 3.60 g/L. The added ferulic acids are consumed completely, wherein most of them (82%) are converted into vanillic acid, a minor part (2%) is metabolized into methoxyl hydroquinone and no vanillin or vanillin alcohol is produced. Alternatively, the fermentation broth of Streptomyces setonii strain ATCC 25497 is used to transform 0.88 g/L of ferulic acid into 0.332 g/L of vanillic acid after 100 hours of growth of the culture. The second step of the two-step method is to add vanillic acid to fermentation broth of microbial strain Phanerochaete chrysosporium MIC 247, wherein 0.3 g/L of vanillic acid is added sequentially (namely: 0.3 g/l at the end of 3 days of culture, then 0.3 g/l every day), and sterile resin XAD2 (Amberlite) is added in the proportion of 10% (weight/volume) after 3 days+6 h of culture of the fungus (that is to say 6 h after adding vanillic acid). Finally, 1.2 g/L of vanillic acid is reduced to 0.628 g/L of vanillin. The subsequent laboratory scale-up study on fermentor employs P. cinnabarinus MUCL 39533 in the conversion of vanillic acid into vanillin and the concentration of vanillin can reach 1,575 mg/L (Stenielaire C., Lesage-meessen. L., Oddou J. et.al., J of Biosci and Bioengineering. 2000, 89, 223-230). The above method employs two kinds of microbes in the conversion of ferulic acid (precursor) into vanillin, wherein the culturing of two kinds of microbes, the conversion reactions of two substrates and extraction of intermediate products (vanillic acid) are involved. Therefore, the whole production process is long and the production yield is low. Rabenhorst et al. (U.S. Pat. No. 6,133,003) discloses the cultivation of Amycolatopsis sp. DSM 9992, wherein ferulic acid is added to the culture in a stepwise manner. After 47 hours of culture, 7,317 ppm of vanillin is produced and 1,526 ppm of ferulic acid is left. This is a conversion rate of approximately 72% of theory. Muheim et al. (U.S. Pat. No. 6,235,507) discloses the cultivation of S. setonii ATCC 39116, wherein 5-40 g/L of ferulic acid is added to the culture in a stepwise manner when the carbon source (glucose) is almost exhausted after 5-40 hours of culture. The culture (biotransformation) is continued for about 5-50 hours and the accumulated vanillin from ferulic acid amounts to 8-16 g/L. A variety of byproducts such as vanillin alcohol, vanillic acid, guaiacol, p-ethenyl guaiacol and 2-methyl-4-ethylphenol, however, are produced in the above biotransformation, which results in a difficulty in separating and extracting converted product.
In the above-described methods for the production of vanillin from ferulic acid by microbial biotransformation, the sources of substrates employed (ferulic acid and vanillic acid) are not described in detail.
Natural ferulic acid exists in a form of trans-ferulic acid and is a constituent of cell wall of plant. Ferulic acid in the cell wall of rice bran plant mainly exists in a form of arabinose glycoside in waste residue of rice bran of plant cell wall. In addition, ferulic acid also exists in forms of cycloartanol and sterol ferulic acid esters in rice bran crude oil with a content of about 2-3%. The niger obtained after extraction of salad oil from rice bran crude oil is rich in ferulic acid esters with a content of about 70%, wherein ferulic acid amounts to up to 25-30% of the ferulic acid esters.